Novel amino acid derivatives

ABSTRACT

Compounds of the formula: &lt;CHEM&gt; (wherein R&lt;1&gt; is hydrogen or alkyl; R&lt;2&gt; is an optionally substituted carbocyclic or heterocyclic ring; Z is hydrogen or a group -COR&lt;9&gt;, (R&lt;9&gt; is alkyl or phenyl) &lt;CHEM&gt; in which R&lt;1&gt;&lt;0&gt; is hydrogen, halogen, trihalomethyl, alkyl or alkoxy, R&lt;1&gt;&lt;1&gt; and R&lt;1&gt;&lt;2&gt; are each hydrogen, alkyl or phenyl substituted by R&lt;1&gt;&lt;0&gt;, or R&lt;1&gt;&lt;1&gt; and R&lt;1&gt;&lt;2&gt; together represent alkylene and p is 1 or 2; q is 1 to 4; A is phenyl or cycloalkyl optionally substituted by halogen, trihalomethyl, alkyl or alkoxy; and m is zero and n is 1 to 4 or n is zero and m is 1 to 4; and non-toxic salts thereof, have an inhibitory effect on enkephalinase and are useful as analgesic, antianxiety and anticonvulsant agents.

SUMMARY

This invention relates to novel amino acid derivatives having aninhibitory activity on enkephalinase. More particularly, it relates toamino acid derivatives of the general formula: ##STR3## (wherein thevarious symbols are as hereinafter described) and non-toxic saltsthereof, which have an inhibitory activity on enkephalinase and whichare, therefore, useful as analgesic against various pain, the processfor the preparation of the derivative of the general formula (I) andenkephalinase inhibitors containing, as active ingredient, thederivative of the general formula (I) or a non-toxic salt thereof.

BACKGROUND

"Enkephalin" is the general term referring to Met⁵ -enkephalin (A is Metin the below formula) or Leu⁵ -enkephalin (A is Leu in the belowformula) of the following formula:

    Try--Gly--Gly--Phe--A

(wherein A represents Met or Leu). These two pentapeptides bind toopioid receptors and such produces analgic effect. They areneurotransmitters (see Nature, 258. 577 (1975)).

Enkephalinase, found by Malfroy et al, is an enzyme which cleavesMet-enkephalin or Leu-enkephalin at the Gly³ -Phe⁴ bond (see Nature,276. 523 (1978)). It plays an important role in the termination of ananalgic effect which enkephalin has.

Accordingly, it is considered that the inhibition for enkephalinaseslows down the deactivation of enkephalin and that the analgic effectmaintains.

PRIOR ARTS

Based on the above fact, recent research and development onenkephalinase inhibitors have been actively carried out.

For example, in European Patent Publication No. 38758, the compounds ofthe general formula: ##STR4## [wherein Xa--Ya represents a mercaptogroup etc.,

na represents zero or one,

Aa--Ba represents a CONH group etc.,

R_(1a) represents a hydrogen atom, an (substituted) alkyl group, a(substituted) phenyl group etc.,

R_(2a) represents a hydrogen atom, an alkyl group, a phenyl group, a(substituted) benzyl group, a hydroxyalkyl group, an (substituted)alkoxyalkyl group, a phenoxyalkyl group or a mercaptoalkyl group,

R_(3a) represents a group of the formula: OR_(4a), NHR_(4a) orN(R_(4a))₂ and

R_(4a) represents a (substituted) phenyl etc.) are proposed. Especially,thiorphan having the formula: ##STR5## is noted (see Nature, 288, 286(1980)).

Thereafter, compounds wherein the glycine moiety in thiorphan isreplaced by various substitutents, have been proposed.

For example, in European Patent Publication No. 136883 (and JapanesePatent Kokai No. 60-136554), the compounds of the general formula:##STR6## (wherein R_(1b) represents a hydrogen atom or ##STR7## R_(2b)represents a group of the formula: ##STR8## R_(3b) represents a hydrogenatom, an alkyl group, a group of the formula: ##STR9## or --(CH₂)_(pb)-cycloalkyl, R_(4b) represents a hydroxy group, an alkoxy group, a groupof the formula: ##STR10## nb represents an integer of 1 to 15) areproposed.

In South African Patent Publication No. 840670, the compounds of thegeneral formula: ##STR11## (wherein R_(1c) represents a hydrogen atom,an acyl group or an aroyl group,

R_(2c) represents a hydrogen atom, an alkyl group, an alkenyl group, analkynyl group or aralkyl group,

R_(3c) represents a hydrogen atom, an alkyl group, a carboxy group, acarboxyamido group, a substituted alkyl group, a substituted aryl group,a thiol group, an alkylthio group or a heteroaryl group,

R_(4c) represents a group of the formula ##STR12## nc represents aninteger of 1 to 3, R_(5c) represents a hydroxy group, an alkoxy group,an aryloxy group, an aralkyloxy group, an NH₂ group, or a group of theformula: ##STR13## are proposed.

In the U.S. Pat. No. 4401677, the compounds of the general formula:##STR14## (wherein R_(1d) represents an alkyl group, a benzyl group or aphenethyl group,

R_(2d) represents an alkyl group or a group of the formula: ##STR15## ndrepresents an integer of 1 to 4) are proposed.

Further, in European Patent Publication No. 254032, the compounds of thegeneral formula: ##STR16## (wherein R_(1ae) represents a phenyl groupsubstituted by one or more substituents independently selected fromalkyl, alkoxy, cycloalkyl etc.

R_(2e) represents a group of the formula:

R_(13e) CONH(CH₂)_(qe) --, R_(13e) NHCO(CH₂)_(qu) --, R_(6e)OCO(CH₂)_(qe--) etc.,

R_(3e) represents a group of the formula: --OR_(7e), --NR_(7e) R_(3e)etc.,

R_(13e) represents a group of the formula: Y_(1e) --C₆ H₄ -- etc.,

R_(6e), R_(7e) and R₈ e represent independently a hydrogen atom, analkyl group, an arylalkyl group etc.,

ne represents zero or an integer 1 or 2,

qe represents an integer 1 to 4,

Qe represents a hydrogen atom or a group of the formula: R_(10e) CO--,

R_(10e) represents an alkyl group, a group of the formula:

Y_(3e) --C₆ H₄ -- etc.,

Y_(1e) and Y_(3e) represent independently a hydrogen atom, an alkylgroup, cycloalkyl group, an alkoxy group etc.) are proposed.

OBJECTS

Energetic investigation has been carried out in order to discovercompounds having an inhibitory effect on enkephalinase, and the presentinventors have found that derivatives wherein

(1) a glycine moiety in thiorphan is replaced by an acidic o-amino acide.g. aspartic acid, glutamic acid etc.) and further

(2) either carboxyl group of the said acidic amino acids is convertedinto an amido bond with various aromatic amines,

have an inhibitory effect on enkephalinase, and have accomplished thepresent invention.

Accordingly, the present invention relates to the amino acid derivativesof the general formula: ##STR17## (wherein R¹ represents a hydrogen atomor an alkyl group of 1 to 4 carbon atoms,

R² represents a carbocyclic or heterocyclic ring, unsubstituted orsubstituted by 1 to 3 R³ s,

R^(s) represents independently:

(1) a halogen atom,

(2) a trihalomethyl group,

(3) a hydroxy group,

(4) an alkyl group of 1 to 15 carbon atoms,

(5) an alkoxy group of 1 to 4 carbon atoms,

(6) an alkylthio, alkylsulfinyl or alkylsulfonyl group, of 1 to 4 carbonatoms,

(7) a group of the formula: ##STR18## in which R⁴ represents a hydrogenatom, a halogen atom, a trihalomethyl group, a hydroxy group, an alkylgroup of 1 to 4 carbon atoms or an alkoxy group of 1 to 4 carbon atoms

(8) a group of the formula:

    --NR.sup.5 R.sup.6

in which R⁵ and R⁶ independently represent a hydrogen atom or an alkylgroup of 1 to 4 carbon atoms,

(9) a group of the formula:

    --CO--R.sup.7

in which R⁷ represents an alkyl group of 1 to 4 carbon atoms or a phenylgroup substituted by R⁴ (in which R⁴ is as hereinbefore defined),

(10) a group of the formula:

    --COOR.sup.8

in which R⁸ represents a hydrogen atom or an alkyl group of 1 to 4carbon atoms,

(11) a group of the formula:

    --CONR.sup.5 R.sup.6

in which R⁵ and R⁶ are as hereinbefore defined,

(12) a group of the formula:

    --SO.sub.2 NR.sup.5 R.sup.6

in which R⁵ and R⁶ are as hereinbefore defined,

(13) a cyano group,

(14) a nitro group, or

(15) a group of the formula:

    --NHCO--R.sup.7,

in which R⁷ is as hereinbefore defined,

Z represents:

(1) a hydrogen atom,

(2) a group of the formula:

    --COR.sup.9

which R⁹ represents an alkyl group of 1 to 4 carbon atoms or a phenylgroup substituted by R¹⁰, in which R¹⁰ represents a hydrogen atom, ahalogen atom, a trihalomethyl group, an alkyl group of 1 to 4 carbonatoms or an alkoxy group of 1 to 4 carbon atoms,

(3) a group of the formula: ##STR19## in which R¹⁰ is as hereinbeforedefined, (4) a group of the formula: ##STR20## in which R¹¹ and R¹²independently represent a hydrogen atom, an alkyl group of 1 to 4 carbonatoms or a phenyl group substituted by R¹⁰, in which R¹⁰ is ashereinbefore defined, or R¹¹ and R¹² together represent an alkylenegroup of 4 or 5 carbon atoms and p is an integer of 1 or 2,

(5) a group of the formula: ##STR21## in which two R¹⁰ are independentlyas hereinbefore defined, (6) a group of the formula: ##STR22## in whichR¹⁰ is as hereinbefore defined and q is an integer of 1 to 4,

(7) a group of the formula: ##STR23## in which R¹⁰ is as hereinbeforedefined, ○A represents a phenyl or cycloalkyl group of 4 to 7 carbonatoms, substituted by R¹³, in which R¹³ represents a hydrogen atom, ahalogen atom, a trihalomethyl group, an alkyl group of 1 to 4 carbonatoms or an alkoxy group of 1 to 4 carbon atoms, and

m and n represent:

(1) when m is zero, n is an integer of 1 to 4, and

(2) when n is zero, m is an integer of 1 to 4), or non-toxic salts ornon-toxic acid addition salts thereof, the process for the preparationof the derivatives of the general formula (I), and enkephalinaseinhibitors containing, as active ingredient, the derivatives of thegeneral formula (I) or non-toxic salts thereof.

The derivatives of the general formula (I) can be classified into twogroups, i.e. α-amide derivatives of the general formula: ##STR24##(wherein the various symbols are as hereinbefore defined) andderivatives introduced an amido bond into β-position or after, of thegeneral formula: ##STR25## (wherein the various symbols are ashereinbefore defined).

There is no description of the compounds of the general formula (Ia) inany of the prior art including the general formulae (a) to (e) mentionedabove, and therefore, the compounds of the general formula (Ia) arenovel.

A part of the compounds of the general formula (Ib) are broadlydisclosed in European Patent Publication No. 254032 (the general formula(e) hereinbefore described). However, the description in the saidEuropean Patent Publication is very broad and in such descriptions, nocompounds are disclosed which had introduced an amido bond into theβ-position or after nor were any compounds practically prepared. Furtherthe biological activity of the compounds was not confirmed. The presentapplicants have synthesized many compounds within the general formula(Ib) and confirmed that they have a useful biological activity.

In the general formula (1), the alkyl groups of 1 to 4 carbon atoms,represented by R¹, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹² and R¹³ aremethyl, ethyl, propyl and butyl groups, and isomers thereof.

The alkoxy groups of 1 to 4 carbon atoms, represented by R³, R⁴, R¹⁰ andR¹³ are methoxy, ethoxy, propoxy and butoxy groups, and isomers thereof.

The alkylthio groups of 1 to 4 carbon atoms, represented by R³ aremethylthio, ethylthio, propylthio and butylthio groups, and isomersthereof, and the alkylsulfinyl groups of 1 to 4 carbon atoms aremethylsulfinyl, ethylsulfinyl, propylsulfinyl and butylsulfinyl groups,and isomers thereof, and further the alkylsulfonyl groups of 1 to 4carbon atoms are methylsulfonyl, ethylsulfonyl, propylsulfonyl andbutylsulfonyl groups, and isomers thereof.

The alkyl groups of 1 to 15 carbon atoms, represented by R³ are methyl,ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl,undecyl, dodecyl, tridecyl, tetradecyl and pentadecyl groups, andisomers thereof.

The halogen atoms represented by R³, R⁴, R¹⁰ and R¹³ are fluorine,chlorine, bromine and iodine atoms, and the trihalomethyl groupsrepresented by R³, R⁴, R¹⁰ and R¹³ are trifluoromethyl, trichloromethyl,tribromomethyl and triiodomethyl groups.

The alkylene group of 4 or 5 carbon atoms, represented by R¹¹ and R¹²together, are tetramethylene or pentamethylene group.

In the general formula (I), the carbocyclic rings represented by R² meanmono-, bi- or tri-cyclic aromatic carbocyclic rings containing not morethan 15 carbon atoms, which may be partially or fully saturated.

Examples of the rings mentioned above are benzene, naphthalene, indene,azulene, fluorene, phenanthrene, anthracene, acenaphthalene, biphenylenerings and partially of fully saturated rings thereof.

More preferably, the carbocyclic ring is mono-, bi- or tri-cyclicaromatic ring composed of benzene skeletons, i.e. benzene, naphthalene,phenanthrene and anthracene.

In the general formula (I), the heterocyclic rings represented by R²mean mono-, bi- or tri-aromatic heterocyclic rings containing not morethan 15 carbon and hetero atoms which may be partially or fullysaturated.

Examples of the rings mentioned above are furan, thiophene, pyrrole,oxazole, isoxazole, thiazole, isothiazole, imidazole, pyrazole, furazan,pyran, pyridine, pyridazine, pyrimidine, pyrazine, indole, isoindole,benzofuran, benzothiophene, indolizine, benzimidazole, benzthiazole,benzoxazole, chromene, quinoline, isoquinoline, quinolizine, purine,indazole, quinazoline, cinnoline, quinoxaline, phthalazine, pteridine,benzodiazepine carbazole, acridine, phenanthridine, xanthene, phenazine,phenothiazine rings and partially or fully saturated rings thereof.

More preferably, the heterocyclic ring is mono-ring or bi-ring condensedwith a benzene ring, containing one or two nitrogen and/or sulfur atoms.

Especially preferred rings represented by R2 are benzene, naphthalene,furan, thiophen, pyridine, pyrimidine, pyrazine, benzimidazole,benzthiazole, benzoxazole and benzodiazepine rings and partiallysaturated rings thereof.

In the general formula (I), the cycloalkyl groups of 4 to 7 carbonatoms, represented by ○A are cyclobutyl, cyclopentyl, cyclohexyl andcycloheptyl groups.

When n is zero, ○A preferably represents a phenyl group or cycloalkylgroup of 4 to 7 carbon atoms, each of which is substituted by R¹³ (R¹³is as hereinbefore defined) and when m is zero, ○A preferably representsan unsubstituted phenyl group, or cycloalkyl group of 4 to 7 carbonatoms which is substituted by R¹³ (R¹³ is as hereinbefore defined).

In the general formula (I), the amino acid residue represented ##STR26##is preferably L-amino acid residue.

The compounds of the general formula (I), of the present invention maybe converted into the corresponding salts by known methods. Non-toxicand water-soluble salts are preferable. Suitable salts are salts ofalkaline metal (sodium, potassium etc.), salts of alkaline earth metal(calcium, magnesium etc.), ammonium salts, salts of pharmaceuticallyacceptable organic amine (tetramethylammonium, triethylamine,methylamine, dimethylamine, cyclopentylamine, benzylamine,phenethylamine, piperidine, monoethanolamine, diethanolamine,tris(hydroxymethyl)aminomethane, lysine, arginine, N-methyl-D-glucamineetc.).

The compounds of the general formula (I), if desired, may be convertedinto acid addition salts by the known methods. Preferably, acid additionsalts are non-toxic salts and water-soluble. The suitable acid additionsalts are, for example, salts of an inorganic acid such as hydrochloricacid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid,nitric acid, or an organic acid such as acetic acid, lactic acid,tartaric acid, benzoic acid, citric acid, methanesulphonic acid,ethanesulphonic acid, benzenesulphonic acid, toluenesulphonic acid,isethionic acid, glucuronic acid and gluconic acid.

The compounds of the general formula (I) can be named as derivatives ofan amino acid. For example, the compound of the formula: ##STR27## canbe called N-(3-mercapto-2-benzylpropionyl)-α-(4-chloroanilino) glutamicacid γ-sodium salt, and the compound of the formula: ##STR28## can becalled N-(3-mercapto-2-benzylpropionyl)aspartic acid β-(1-naphthyl)amideα-methyl ester.

Throughout the specification including claims, it may be easilyunderstood by those skilled in the art, that the alkyl, alkoxy,alkylthio, alkylsulfinyl and alkylsulfonyl groups includestraightchained and also branched-chained ones.

Accordingly, throughout the specification including claims, all isomersproduced by the difference in stereo configuration, such as asymmetriccarbons are included in the present invention.

According to the present invention, the compounds of the general formula(I), of the present invention, may be prepared by using a series ofreactions depicted in Scheme A, B and C below, wherein

R^(1') represents an alkyl group of 1 to 4 carbon atoms,

R¹⁴ represents a silyl group substituted by three substituents which areselected from an alkyl group of 1 to 4 carbon atoms and a phenyl group(e.g. a tert-butyldimethylsilyl, diphenyl-tert-butylsilyl group),

Z¹ represents a group of the formula: --COR⁹ (in which R⁹ is ashereinbefore defined),

Z² represents the group other than a hydrogen atom in the groupsrepresented by Z (Z is as hereinbefore defined),

m₁ and n₁ represent:

(1) when m1 is zero, n1 is an integer of 1 to 4, and

(2) when n1 is zero, m1 is an integer of 2 to 4,

^(t) Bu represents a tert-butyl group,

Bn represents a benzyl group, and

the other symbols are as hereinbefore defined. ##STR29##

Each of the steps depicted in the said schemes, are well known to thoseskilled in the art. For example, Reaction 1 may be carried out byreacting an amine of the formula (II) with a carboxylic acid of theformula: ##STR30## (wherein the various symbols are as hereinbeforedefined) to form an amido bond. The said reactions are known and, forexample are

(A) method using a mixed acid anhydride,

(B) method using an acid halide,

(C) method using a condensing agent such as DCC etc.

DETAILED DESCRIPTION OF THE REACTIONS

The foregoing reactions (A), (B) and (C) are described in greaterdetailed wherein:

(A) is a method using a mixed acid anhydride which may be carried out,for example, by reacting carboxylic acid of the general formula (VI) andan acid halide (pivaloyl chloride, tosyl chloride, mesyl chloride etc.)or an acid derivative (ethyl chloroformate, isobutyl chloroformate etc.)in the presence of a tertiary amine (pyridine, triethylamine, picolineetc.) in an inert organic solvent (chloroform, methylene chloride,diethyl ether, THF etc.) or without a solvent at a temperature of from0° C. to 40° C., and then reacting the obtained mixed acid anhydridewith an amine of the general formula (II) in an inert organic solvent(as hereinbefore described), at a temperature of from 0° C. to 40° C.,

(B) is a method using an acid halide which may be carried out, forexample, by reacting a carboxylic acid of the general formula (VI) withan acid halide (thionyl chloride, oxalyl chloride etc.) in an inertorganic solvent (as hereinbefore describe) or without a solvent at from-20° C. to the reflux temperature of a solvent, and then reacting theobtained acid halide with an amine of the general formula (II) in thepresence or absence of a tertiary amine (as hereinbefore described) inan inert organic solvent (as hereinbefore described), at a temperatureof from 0° C. to 40° C., and

(C) is a method using a condensing agent such as DCC(dicyclohexylcarbodiimide) etc. which may be carried out, for example,by reacting a carboxylic acid of the general formula (VI) with an amineof the general formula (II) using DCC in the presence or absence of atertiary amine (as hereinbefore described), in an inert organic solvent(as hereinbefore described) or without a solvent, at a temperature offrom 0° C. to 40° C.

The reactions of (A), (B) and (C) hereinbefore described are preferablycarried out in an atmosphere of inert gas (argon, nitrogen etc.) underanhydrous conditions.

Reaction 2 may be carried out by reacting with 2-mercaptoethylamine(HS--(CH₂)₂ --NH₂) in an inert organic solvent (methylene chloride,acetonitrile etc.), at a temperature of from ambient to 60° C.

Reaction 3 may be carried out by reacting with anhydrous potassiumcarbonate or anhydrous sodium carbonate in an absolute alkanol (e.g.absolute methanol, absolute ethanol etc.), ordinarily at a temperatureof from -10° C. to 100° C.

Reaction 4 may be carried out by using an aqueous solution of an alkali(potassium hydroxide, sodium hydroxide, lithium hydroxide, potassiumcarbonate, sodium carbonate etc.) in a water-miscible organic solvent(dimethoxyethane, THF (tetrahydrofuran), dioxane, lower alkanol amine ofthe general formula (II) in an inert organic solvent (as etc.),ordinarily at a temperature of from -10° C. to 100° C.

Reaction 5 is a hydrolysis under acidic conditions, and may be carriedout by reacting with an aqueous solution of an organic acid (aceticacid, oxalic acid, p-toluenesulfonic acid, trifluoroacetic acid etc.) orwith an aqueous solution of an inorganic acid (hydrochloric acid,sulfuric acid etc.) in an inert organic solvent (e.g. methylenechloride, THF, dioxane, a lower alkanol etc), at a temperature of fromambient to a reflux temperature of a solvent.

Reaction 6 may be carried out by reacting with anhydrous potassiumcarbonate or anhydrous sodium carbonate in an absolute alkanolcorresponding to the desirable R^(1a) (absolute methanol or absoluteethanol etc.), ordinarily at a temperature of from -10° C. to 100° C.

Reaction 7, when Z² is a substituted acyl group (the groups of (2)˜(6)in those represented by Z), may be carried out by reacting with a halideor acid anhydride of the carboxylic acid corresponding to Z², in thepresence of a tertiary amine (pyridine, triethylamine etc.), in an inertorganic solvent (methylene chloride, dimethylformamide, THF, ethylacetate etc.) or in the absense of a solvent, at a temperature of from0° C. to 50° C., or when Z² represents a group of the formula: ##STR31##(in which R¹⁰ is as hereinbefore defined), may be carried out byreacting with a halide of the compound represented by Z², in thepresence of a base (a tertiary amine, a hydroxide or carbonate, of analkali metal etc.), in an inert organic solvent (methylene chloride,dimethylformamide, THF, acetone etc.) at a temperature of from 10° C. to50° C.

Reaction 8 is a desilylation, and may be carried out by usingtetrabutylammonium fluoride (_(n) B_(u4) N⁺ F³¹) in tetrahydrofuran atroom temperature.

Reaction 9 is an esterification, and may be carried out by reacting witha desirable diazoalkane (diazomethane etc.), in an inert organic solvent(diethyl ether, ethyl acetate, methylene chloride, acetone, a loweralkanol etc.) at a temperature of from -10° C. to 40° C., or by reactinga desirable alkanol in the presence of an acid (hydrochloric acid,p-toluenesulfonic acid etc.) or in the presence of a condensing agent(dicyclohexylcarbodiimide etc.), at a temperature of from -10° C. to 50°C.

Reaction 10 may be carried out by the same procedure as Reaction 7 whenZ² represent a group of the formula: ##STR32##

The compounds of the general formulae (II), (III), (IV) and (V), used inthe aforesaid schemes, may be prepared by the combination of knownmethods, for example, by using a series of reactions depicted in thefollowing Scheme D, wherein Boc represents a tert-butoxycarbonyl group,cbz represents a benzyloxycarbonyl group and the other symbols are ashereinbefore defined. ##STR33##

Each of the steps depicted in the said scheme are well known to thoseskilled in the art.

Throughout the specification, in each reactions, products may bepurified by conventional methods, for example, distillation atatmospheric or reduced pressure, high performance liquid chromatography,thin layer chromatography or column chromatography using silica gel ormagnesium silicate or washing or recrystallization. Purification may becarried out after each reaction, or after a series of reactions.

Starting materials of the formula (VII) and (IX) and every reagents,used in the process for the preparation, of the present invention, areknown compounds per se. or may be easily prepared by known methods.

For example, the compounds of the formula (VII), wherein n is zero and mis 1 or 2, that of the formula (IX), wherein n1 is zero and m₁ is 1 or2, and that of the formula (IX), wherein m: is zero and n1 is 1 or 2,are on the market.

The compounds of the general formula (I) or non-toxic salts thereof, ofthe present invention have an inhibitory effect on enkephalinase, andare, therefore, useful as analgesic, antianxiety agents oranticonvulsant, in mammals, especially in humans.

The inhibitory effect on enkephalinase and analgesic effect based on theinhibitory effect, of the compounds of the present invention wereconfirmed by screening tests as described below.

Inhibitory effect on enkephalinase (1) Method

Enkephalinase was obtained by the procedure as described in Journal ofNeurochemistry, 39. 1081 (1982). That is, to striata obtained from ddYmale mice (weighing 28˜30 g), Tris hydrochloric acid buffer solution(referred to "Tris buffer" hereafter) was added. The mixture washomogenised and centrifuged (1000 g ×5 minites). The supernatantobtained was further centrifuged (20000 g ×1 minutes). The resultingpellet was washed with cold Tris buffer and resuspended in a fresh Trisbuffer to use as enzyme source of enkephalinase.

The experiment was carried out according to the method as described inJournal of Biological Chemistry, 255. 2227 (1980). That is, the testcompounds (10⁻⁵ ˜10⁻⁹ M) were dissolved in 2% DMSO/Tris buffer (50 μl).Incubation at 37° C. for 60 minutes was started by adding thereto 150 μgof enzyme solution (×30 dilution of the above enzyme source) and 50 μlof a substrate solution containing succinyl-alanylalanyl-phenylalanyl(7-amido-4-methyl)coumarin (i.e. Suc Ala-Ala-Phe-AMC, finalconcentration: 10⁻⁴ M) dissolved in 50 mM HEPES/NaOH buffer (pH 7.4).The reaction was stopped by the addition of thiorphan (10⁻⁶ M) and byheating the samples at 95° C for 15 minutes. In a second step, theincubation medium was further incubated at 56° C for 60 minutes in thepresence of 0.75 μl of aminopeptidase M. The appearance of AMCfluorescence was measured (exc. 367 nm, em. 440 nm). Blanks values wereobtained by the same procedure as above described by using thiorphan(10⁻⁶ M) instead of the test compound.

(2) Result

The results are shown in Table 1 below.

                  TABLE I                                                         ______________________________________                                        Inhibitory effect on enkephalinase (1)                                         ##STR34##                                                                    Compounds                      Inhibitory                                     Example  Structure             effect                                         No.      R.sup.1                                                                              R.sup.2          m   (IC.sub.50, M)                           ______________________________________                                        1        H                                                                                     ##STR35##       2   2.1 × 10.sup.-8                    8(4)     H                                                                                     ##STR36##       1   5.0 × 10.sup.-8                    3(2)     H                                                                                     ##STR37##       2   3.6 × 10.sup.-9                    3(8)     H                                                                                     ##STR38##       2   2.3 × 10.sup.-8                    2(7)     H                                                                                     ##STR39##       2   3.5 × 10.sup.-7                    3(4)     H                                                                                     ##STR40##       2   3.5 × 10.sup.-8                    3(9)     H                                                                                     ##STR41##       2   1.9 × 10.sup.-7                     2(10)   H                                                                                     ##STR42##       2   4.4 × 10.sup.-8                    4        CH.sub.3                                                                              ##STR43##       1   1.5 × 10.sup.-7                    ______________________________________                                    

                  TABLE I                                                         ______________________________________                                        Inhibitory effect on enkephalinase (2)                                         ##STR44##                                                                    Compounds                      Inhibitory                                     Example  Structure             effect                                         No.      R.sup.1                                                                              R.sup.2          n   (IC.sub.50, M)                           ______________________________________                                        12       Na                                                                                    ##STR45##       1   1.3 × 10.sup.-8                    11       Na                                                                                    ##STR46##       2   6.8 × 10.sup.-8                    12(5)    H                                                                                     ##STR47##       1   2.8 × 10.sup.-9                    12(7)    H                                                                                     ##STR48##       1   2.4 × 10.sup.-8                    12(8)    H                                                                                     ##STR49##       1   2.1 × 10.sup.-8                    ______________________________________                                    

                  TABLE I                                                         ______________________________________                                        Inhibitory effect on enkephalinase (3)                                         ##STR50##                                                                    Compounds                 Inhibitory                                          Example    Structure      effect                                              No.         ○A     (IC.sub.50, M)                                      ______________________________________                                        1(1)                                                                                      ##STR51##     4.0 × 10.sup.-7                               1(2)                                                                                      ##STR52##     5.1 × 10.sup.-8                               1(3)                                                                                      ##STR53##     7.1 × 10.sup.-8                               ______________________________________                                    

Inhibitory effect on bradykinin-induced bitinq-like response

(1) Method

The experiment was carried out according to the method as described inJournal of Pharmacological Methods, 7, 271 (1982). That is, maleSpraque-Dawley rats (weighing 200˜300 g) were used. Implantation of thebradykinin (0.63˜1.25 μg in 0.5˜1.0 μl of distilled water)--filledcannula onto the tooth pulp and the fixation of it on the lower incisorsurfaces were carried out under ethyl ether anesthesia. Amicro-application of bradykinin onto the tooth pulp produced biting-likeresponse and some other aversive behaviors such as jumping, struggling,rubbing, scratching, escape, head-jerk and body-jerk within 1 minute.Only rats which showed a duration of 20 minutes or more for thebiting-like responses before administration of the compound of thepresent invention, were used for further experiment.

The test compounds suspended in 0.5 % carboxymethyl cellulose solutionwere administered either intraperitoneally or orally. After regularintervals bradykinin was administered. The number of rats which did notshow the biting-like response after administration (i.e. analgic state)was counted.

(2) Result

The results are shown in Table II below.

                                      TABLE II                                    __________________________________________________________________________    Inhibitory effect on bradykinin-induced biting-like response                                                                  Number                        Com-                                        Num-                                                                              of rats which did not                                                         show                          pounds                                      ber the biting-like response      Example                                Dose of  10                                                                              40                                                                              70                                                                              100 minutes (i.p.)      No.  Structure                         (mg/kg)                                                                            rats                                                                              30                                                                              60                                                                              90                                                                              120 after               __________________________________________________________________________                                                          (p.o.)                        ##STR54##                        10 (i.p.)                                                                          5   4 4 3 1                       12(5)                                                                               ##STR55##                        10 (i.p.)                                                                          8   4 2 1 0                       16                                                                                  ##STR56##                        100 (p.o.)                                                                         8   3 6 2 0                       13(5)                                                                               ##STR57##                        100 (p.o.)                                                                         8   1 7 1 1                       __________________________________________________________________________

On the other hand, it was confirmed that the acute toxicity of thecompound of the present invention were very weak. For example, acutetoxiety (LD₅₀) ofN-3-(phthalid-3-yl)thio-2S-benzylpropionyl-α-anilino-L-gluatamic acid is500˜1000 mg/kg in intraperitoneal injection in mice. Therefore, thecompounds of the present invention may be considered to be sufficientlysafe and suitable for pharmaceutical used.

For the purpose above described, the compounds of the present inventionmay normally be administered systemically or partially, usually by oralor parenteral administration.

The doses to be administered are determined depending upon age, bodyweight, symptom, the desired therapeutic effect, the route ofadministration, and the duration of the treatment etc. In the humanadult, the doses per person per dose are generally between 10 mg and 1g, by oral administration, up to several times, preferably 1 to 4 times,per day, and between 1 mg and 100 mg, by parenteral administration(preferably, intravenous administration) up to several times, preferably1 to 4 times, per day.

As mentioned above, the doses to be used depend upon various conditions.Therefore, there are cases in which doses lower than or greater than theranges specified above may be used.

The compounds of the present invention were administered as solidcompositions, liquid compositions and other compositions for oraladministration and injections, external compositions and suppositoriesetc. for parenteral administration.

Solid compositions for oral administration, include compressed tablets,pills, capsules, dispersible powders, and granules. In suchcompositions, one or more of the active compounds are admixed with atleast one inert diluent (lactose, mannitol, glucose,hydroxypropylcellulose, microcrystalline cellulose, starch,polyvinylpyrrolidone, magnesium metasilicate aluminate etc.).

The compositions may also comprise, as is normal practice, additionalsubstances other than inert diluents, e.g. lubricating agents (magnesiumstearate etc.), disintegrating agents (cellulose calcium glycolateetc.), and assisting agent for dissolving (glutamic acid, aspertic acidetc.) and stabilizing agent (lactose etc.).

The tablets or pills may, if desired, be coated with film of gastric orenteric material (sugar, gelatin, hydroxypropylcellulose,hydroxypropylmetnyl cellulose phthalate etc.).

The term "capsules" include both soft and hard ones.

Liquid compositions for oral administration includepharmaceutically-acceptable solutions, emulsions, suspensions, syrupsand elixirs.

In such liquid compositions, one or more of the active compounds areadmixed with inert diluents commonly used in the art (purified water,ethanol etc.).

Besides inert diluents, such compositions may also comprise adjuvantssuch as wetting agents and suspending agents, sweetening agents,flavouring agents, perfuming agents and preserving agents.

Other compositions for oral administration include spray compositionswhich may be prepared by known methods and which comprise one or more ofthe active compounds. Spray compositions may comprise additionalsubstances other than inert diluents, e.g. stabilizing agents (sodiumsulfite etc.), isotonic buffer (sodium chloride, sodium citrate, citricacid etc.). For preparation of such spray compositions, for example, themethod described in the U.S. Pat. No. 2868691 or 3095355 may be used.

Injections for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions and emulsions.

In such injections, one or more of active compounds are admixed with atleast one of inert aqueous diluents (distilled water for injection,physiological salt solution etc.) or inert non-aqueous diluents(propylene glycol, polyethylene glycol, olive oil, ethanol, POLYSOLBATE80 (registered trade mark) etc.).

Injections may comprise the addition of other than inert diluents, e.g.preserving agents, wetting agents, emulsifying agents, dispersingagents, stabilizing agents (lactose etc.), assisting agents such asassisting agents for dissolving (glutamic acid, aspertic acid etc.).

They may be usually sterilized by filtration (through abacteria-retaining filter etc.), incorporation of sterilizing agents inthe compositions or by irradiation. After sterilizing as described, theyalso be manufactured in the form of sterile solid compositions, forexample, by freeze-drying, and which can be dissolved in sterile wateror some other sterile diluents for injection immediately before used.

Other compositions for parenteral administration include liquids forexternal use, and endermic liniments (ointment etc.), suppositories andpessaries which comprise one or more of the active compounds and may beprepared by known methods.

The following reference examples and examples illustrate, but not limit,the present invention.

The solvents in the parentheses show the eluting or developing solventsand the ratios of the solvents used are by volume in chromatographicseparations. Unless otherwise specified, "IR" was measured by KBr methodand "NMR" was measured in deuterochloroform (CDCl₃) solution.

REFERENCE EXAMPLE 1 N-(tert-butoxycarbonyl)-α-anilino-L-glutamic acidγ-benzyl ester ##STR58##

To a solution of N-(tert-butoxycarbonyl)-L-glutamic acid γ-benzyl esterdicyclohexylamine salt (being on a market, 5.18 g) in methylene chloride(30 ml) was added pivaloyl chloride (1.35 ml) under cooling with ice,and the mixture was stirred for 10 minutes at room temperature. Thereaction mixture was again cooled with ice, and then a solution ofaniline (1 ml) in triethylamine (1.4 ml) was added dropwise thereto, andthe mixture was stirred for 20 minutes at room temperature. The reactionmixture was diluted with ethyl acetate, washed with 1N hydrochloricacid, IN aqueous solution of sodium hydroxide and a saturated aqueoussolution of sodium chloride, successively, dried over magnesium sulfate,and concentrated under reduced pressure.

The residue (solid) was washed with n-hexane to give the title compound(2.9 g) having the following physical data:

TLC (ethyl acetate : n-hexane =1:1):Rf 0.74;

NMR: δ8.40 (IH, brs), 7.55˜6.90 (1OH, m), 5.35 (IH, brd), 5.10 (2H, s),4.40˜4.10 (1H, m), 2.80˜2.40 (2H, m), 2.40˜1.70 (2H, m),

1.42 (9H, s);

MS:m/z 412(M+), 356, 339, 292.

REFERENCE EXAMPLE 2 α-anilino-L-glutamic acid γ-benzyl estertrifluoroacetic acid salt ##STR59##

To a solution of glutamic acid protected by a Boc group (2.9g, preparedin Reference Example 1) in methylene chloride (2 ml) was addedtrifluoroacetic acid (5.4 ml) under cooling with ice, and the mixturewas stirred for 2.5 hours at a room temperature. The reaction mixturewas concentrated under reduced pressure to give the crude title compound(3.00 g) having the following physical data:

TLC (ethyl acetate) : Rf 0.23;

NMR: δ9.50(IH, brs), 7.60˜6.80(10H, m), 5.00(2H, s), 4.60˜4.30(1H, m),2.70˜2.50(2H, m), 2.50˜1.90(2H, m);

MS:m/z 312, 204, 193, 192.

REFERENCE EXAMPLE 3N-(3-acetylthio-2RS-benzylpropionyl)-α-anilino-L-glutamic acid γ-benzylester ##STR60##

To a solution of 3-acetylthio-2RS-benzylpropionic acid (prepared by themethods described hereafter, 833 mg) in methylene chloride (1 ml) wasadded an excess amount of oxalyl chloride at room temperature, and themixture was stirred for 30 minutes. Oxalyl chloride was fully distilledoff from the reaction mixture, and to the residue was added methylenechloride (2 ml) to obtain a solution of acid chloride.

To a solution of trifluoroacetic acid salt of the amine compound(prepared in Reference Example 2, 1.64 g) in a mixture of pyridine (2.83ml) and methylene chloride (16 ml), was added dropwise the solution ofacid chloride prepared hereinbefore under cooling with ice and themixture was stirred for 15 minutes at room temperature. The reactionmixture was poured into water, washed with 1N hydrochloric acid, 1Naqueous solution of sodium hydroxide and a saturated aqueous solution ofsodium chloride, successively, dried over magnesium sulfate andconcentrated under reduced pressure.

The residue was purified by column chromatography on silica gel(methylene chloride : n-hexane =2 : 1 →methylene chloride→methylenechloride : methanol =20 : 1) and further recrystallized from a mixtureof ethyl acetate and n-hexane to give the title compound (743 mg) aswhite powder having the following physical data:

TLC (ethyl acetate : n-hexane =1 : 1) : Rf 0.48;

NMR: 6 8.52 and 8.38(IH, brs), 7.34˜7.00(15H, m), 6.44˜6.24(IH, m), 5.12and 5.10(2H, s), 4.60˜4.30(IH, m), 3.24˜3.00 and 3.00˜2.82(4H, m),2.76˜2.34(3H, m), 2.28 and 2.22(3H, s), 2.20˜1.82(2H, m);

MS:m/z 532(M⁺), 489, 440, 370.

3-Acetylthio-2RS-benzylpropionic acid, used as a starting material in aprocedure hereinbefore described, was prepared as follows.

To 2RS-benzylacrylic acid (being on the market, 25 g) was addedthioacetic acid (16 ml) and the mixture was refluxed for one hour byheating on oil bath. An excess amount of thioacetic acid was distilledoff from the residue and further distilled off an azeotropic mixturewith toluene. The residue was purified by column chromatography onsilica gel (ethyl acetate : n-hexane : acetic acid=5:95:0.05→20:80:0.05)to give 3-acetylthio-2RS-benzylpropionic acid (16.7 g) as colorless oilhaving the following physical data:

TLC (n-hexane:ethyl acetate=1:1):Rf 0.3;

MMR:δ7.35˜7.14(5H, m), 3.2˜2.8(5H, m), 2.33(3H, s);

MS:m/z 238(M⁺), 220.

EXAMPLE 1 N-(3-mercapto-2RS-benzylpropionyl)-α-anilino-L-gluatmic acidand their γ-sodium salt ##STR61##

Under an atmosphere of argon, to a solution of the benzyl ester compound(prepared in Reference Example 3, 743 mg) in a mixture oftetrahydrofuran (9) ml) and dimethoxyethane (1.4 ml) was added asolution of lithium hydroxide monohydrate (293 mg) in water (5 ml) atroom temperature and the mixture was stirred for 30 minutes at the sametemperature. To the reaction mixture was added 1N hydrochloric acidunder cooling with ice to acidify and the mixture was extracted withethyl acetate. The extract was washed with a saturated aqueous solutionof sodium chloride, dried over sodium sulfate and concentrated underreduced pressure.

The residue was purified by column chromatography on silica gel(chloroform→acetic acid : chloroform =1 : 99) to give the title compound(387 mg as free acid) as white powder having the following physicaldata:

Melting point: 158.5˜160.0 ° C.;

TLC (acetic acid : chloroform =5 : 95) : Rf 0.20;

NMR(CDCl₃ +DMSO-d₆) : δ9.04˜8.86(IH, d like m)), 7.56˜7.40(2H, m),7.36˜6.92(9H, m), 4.72˜4.46(1H, m), 3.02˜2.40 (6H, m), 2.40˜1.68(4H, m),1.54(1H, t) ;

MS:m/z 400(M⁺), 382, 366, 353, 335;

IR(KBr): ν3275, 1700, 1640, 1600, 1540, 1440, 1300, 1250, 750, 695 cm⁻.

The free acid compound obtained above was dissolved in a small amount ofmethanol and IN aqueous solution of sodium hydroxide and water wereadded thereto and the mixture was stirred for 5 minutes at roomtemperature. The reaction mixture was lyophilized to give the titlecompound (as soidum salt) having the following physical data:

TLC (chloroform : acetic acid =95 : 5) : Rf 0.20;

NMR(CD₃ OD): δ7.60˜7.00(1OH, m)), 4.50˜4.20(1H, m), 3.05˜1.70(9H, m);

IR: ν3680˜2500(3270) 1635, 595, 1540, 1440, 1400, 1310, 1250, 750, 695cm⁻.

The desired compounds shown in the following Table III were obtained bythe same procedure as a series of reactions of Reference Example 1→Reference Example 2 →Reference Example 3 →Example 1, by using acorresponding 3-acetylthio-2-substituted propionic acid.

                                      TABLE III                                   __________________________________________________________________________     ##STR62##                                                                    Example                                                                       No.   ○A Chemical name      TLC    IR (ν, cm.sup.-1)                __________________________________________________________________________    1(1)                                                                                ##STR63## N-(3-mercapto-2RS-cyclohexylmethyl- propionyl)-α-ani                    lino-L-glutamic acid                                                                             Rf 0.33 (acetic acid: chloroform =                                            5:95)  3600˜2400, 1710, 1640,                                                  1540, 1440, 1245, 755               1(2)                                                                                ##STR64## N-[3-mercapto-2RS-(4-methoxybenzyl)- propionyl]-α-an                    ilino-L-glutamic acid                                                                            Rf 0.40 (acetic acid: chloroform =                                            1:9)   3650˜2250, (3270, 3040,                                                 2920), 1700˜1600(1630),                                                 1505, 1440, 1295, 1240, 1175,                                                 1115, 750, 690                      1(3)                                                                                ##STR65## N-[3-mercapto-2RS-(4-methylbenzyl)- propionyl]-α-ani                    lino-L-glutamic acid                                                                             Rf 0.47 (acetic acid: chloroform =                                            1:9)   3650˜2200(3270, 3040,                                                   2925), 1705, 1640, 1600, 1540,                                                1440, 1305, 1250, 755               __________________________________________________________________________

EXAMPLE 2

The desired compounds shown in the following Table IV were obtained bythe same procedure as a series of reactions of Reference Example 1→Reference Example 2 (hydrochloric acid instead of trifluoroacetic acidwas used) →Reference Example 3 →Example 1, by using correspondingstarting material and the amine compound.

                                      TABLE IV                                    __________________________________________________________________________     ##STR66##                                                                    Example                                     IR (ν, cm.sup.-1) or           No.  R.sup.2     Chemical name       TLC    Melting point                     __________________________________________________________________________    2(1)                                                                                ##STR67##  N-(3-mercapto-2RS-benzylpropionyl)- α-(4-fluoroanil                     ino)-L-glutamic acid                                                                              Rf 0.19 (acetic acid: chloroform =                                            8:92)  3275, 1705, 1640, 1540, 1505,                                                 1440, 1405                        2(2)                                                                                ##STR68##  N-(3-mercapto-2RS-benzylpropionyl)- α-(4-chloroanil                     ino)-L-glutamic acid                                                                              Rf 0.28 (acetic acid: chloroform =                                            8:92)  3275, 1705, 1640, 1600, 1530,                                                 1495                              2(3)                                                                                ##STR69##  N-(3-mercapto-2RS-benzylpropionyl)- α-(4-iodoanilin                     o)-L-glutamic acid  Rf 0.34 (acetic acid: chloroform =                                            8:92)  181.0˜185.0° C.      2(4)                                                                                ##STR70##  N-(3-mercapto-2RS-benzylpropionyl)-  α-(4-methoxyan                     ilino)-L-glutamic acid                                                                            Rf 0.25 (acetic acid: chloroform =                                            2:8)   3275, 1700, 1640, 1510, 1440,                                                 1420, 1300, 1240                  2(5)                                                                                ##STR71##  N-(3-mercapto-2RS-benzylpropionyl)- α-(4-trifluorom                     ethylanilino)-L- glutamic acid                                                                    Rf 0.28 (acetic acid: chloroform =                                            5:95)  3275, 1700, 1690, 1640, 1610,                                                 1520, 1410                        2(6)                                                                                ##STR72##  N-(3-mercapto-2RS-benzylpropionyl)- α-[4-(N,N-dimet                     hylamino)anilino]-L- glutamic acid                                                                Rf 0.43 (chloroform: methanol:                                                acetic acid = 30:3:1)                                                                3275, 1710, 1650, 1640, 1520,                                                 1450                              2(7)                                                                                ##STR73##  N-(3-mercapto-2RS-benzylpropionyl)- L-glutamic acid                           α-(1-naphthyl)amide                                                                         Rf 0.25 (acetic acid: chloroform =                                            5:95)  3625˜2300(3260, 3025),                                                  1695, 1630, 1525, 1500, 1430,                                                 1395, 1265, 790, 770, 695         2(8)                                                                                ##STR74##  N-(3-mercapto-2RS-benzylpropionyl)- α-(4-methylanil                     ino)-L-glutamic acid                                                                              Rf 0.29 (acetic acid: chloroform =                                            8:92)  3275, 1710, 1660, 1640, 1610,                                                 1540, 1530, 1510, 1440, 1410      2(9)                                                                                ##STR75##  N-(3-mercapto-2RS-benzylpropionyl)- L-glutamic acid                           α-(4-pyridyl)amide                                                                          Rf 0.18 (chloroform: methanol:                                                acetic acid = 30:5:1)                                                                3250, 1720, 1700, 1630, 1590,                                                 1500                               2(10)                                                                              ##STR76##  N-(3-mercapto-2RS-benzylpropionyl)- L-glutamic acid                           α-(2-benzthiazolyl) amide                                                                   Rf 0.52 (chloroform: methanol:                                                acetic acid = 30:3:1)                                                                3650˜2150(3275, 3050,                                                   2925), 1700, 1640, 1530,                                                      1440, 1305, 1285, 750,            __________________________________________________________________________                                                700                           

EXAMPLE 3

The desired compounds shown in the following Table V and VI wereobtained by the same procedure as a series of reactions of ReferenceExample 1 (the amido bond-forming reaction usingdicyclohexylcarbodiimide (DCC) as a condensing agent, was used insteadof the method using a mixed acid anhydride with pivaloylchloride)→Reference Example 2 (hydrochloric acid instead oftrifluoroacetic acid was used) →Reference Example 3 →Example 1, by usingcorresponding starting material and the amine compound.

                                      TABLE V                                     __________________________________________________________________________     ##STR77##                                                                    Example                                                                       No.  R.sup.2      Chemical name     TLC     IR (ν, cm.sup.-1)              __________________________________________________________________________    3(1)                                                                                ##STR78##   N-(3-mercapto-2RS-benzylpropionyl)- α-(4-cyanoanil                      ino)-L-glutamic acid                                                                            Rf 0.36 (acetic acid: chloroform =                                            1:9)    3650˜2320(3300), 2240,                                                  1690, 1640, 1590, 1410, 1310,                                                 1255, 1180, 840, 755, 700,                                                    555                               3(2)                                                                                ##STR79##   N-(3-mercapto-2RS-benzylpropionyl)- α-(4-carboxyan                      ilino)-L-glutamic acid                                                                          Rf 0.39 (chloroform: methanol: acetic                                         acid = 30:3:1)                                                                        3700˜2340(3270, 3050),                                                  1690, 1680, 1640, 1595, 1525,                                                 1410, 1250, 1175, 1005, 855,                                                  775, 700                          3(3)                                                                                ##STR80##   N-(3-mercapto-2RS-benzylpropionyl)- α-(4-acetylani                      lino)-L-glutamic acid                                                                           Rf 0.34 (acetic acid: chloroform =                                            1:9)    3670˜2330(3350), 1710,                                                  1640, 1595, 1525, 1405, 1360,                                                 1320, 1270, 1180, 840, 700        3(4)                                                                                ##STR81##   N-(3-mercapto-2RS-benzylpropionyl)- L-glutamic acid                           α-(5-chloropyridin- 2-yl)amide                                                            Rf 0.41 (acetic acid: chloroform =                                            5:95)   3640˜2200(3300, 3050),                                                  1705, 1640, 1580, 1520, 1460,                                                 1380, 1305, 1115, 700             3(5)                                                                                ##STR82##   N-(3-mercapto-2RS-benzylpropionyl)- α-(4-nitroanil                      ino)-L-glutamic acid                                                                            Rf 0.17 (acetic acid: chloroform =                                            4:96)   3300, 1710, 1645, 1620, 1600,                                                 1500, 1450, 1415, 1340            3(6)                                                                                ##STR83##   N-(3-mercapto-2RS-benzylpropionyl)- α-(4-carbamoyl                      anilino)-L-glutamic acid                                                                        Rf 0.38 (acetic acid: ethyl acetate =                                         :95)    3300, 1710 (shoulder),                                                        1700˜1660 (shoulder),                                                   1640, 1605, 1520, 1410            3(7)                                                                                ##STR84##   N-(3-mercapto-2RS-benzylpropionyl)- α-(4-decylanil                      ino)-L-glutamic acid                                                                            Rf 0.46 (acetic acid: chloroform =                                            5:95)   3280, 2940, 2860, 1700,                                                       1680, 1600, 1515, 1445, 1410,                                                 1255, 700                         3(8)                                                                                ##STR85##   N-(3-mercapto-2RS-benzylpropionyl)- α-(4-sulfamoyl                      anilino)-L-glutamic acid                                                                        Rf 0.52 (acetic acid: ethyl acetate =                                         :95)    3300, 1700 (shoulder), 1645,                                                  1595, 1520, 1400, 1330, 1255,                                                 1160                              3(9)                                                                                ##STR86##   N-(3-mercapto-2RS-benzylpropionyl)- L-glutamic acid                           α-(pyrazin-2-yl) amide                                                                    Rf 0.26 (acetic acid: chloroform =                                            1:9)    3650˜2240(3275, 3040,                                                   2930), 1705, 1630, 1530,                                                      1410, 1300, 1270, 1210, 1060,                                                 1010, 845, 750, 700                3(10)                                                                              ##STR87##   N-(3-mercapto-2RS-benzylpropionyl)- L-glutamic acid                           α-(pyrimidin-2-yl) amide                                                                  Rf 0.26 (chloroform: methanol:                                                (acetic acid = 30;3:1)                                                                3250, 1710, 1650, 1580, 1510,                                                 1440, 1415                         3(11)                                                                              ##STR88##   N-(3-mercapto-2RS-benzylpropionyl)- α-(2-iodoanili                      no)-L-glutamic acid                                                                             Rf 0.24 (acetic acid:  chloroform =                                           5:95)   3250, 3025, 1700, 1630, 1575,                                                 1515, 1430, 1280, 1010, 750,                                                  700                                3(12)                                                                              ##STR89##   N-(3-mercapto-2RS-benzylpropionyl)- α-(3-iodoanili                      no)-L-glutamic acid                                                                             Rf 0.24 (acetic acid: chloroform =                                            5:95)   3260, 3030, 1705, 1635, 1580,                                                 1520, 1470, 1410, 1300, 1240,                                                 865, 780, 750, 700,               __________________________________________________________________________                                                680                           

                                      TABLE VI                                    __________________________________________________________________________     ##STR90##                                                                    Example                                    Optical rotation                   No.  R.sup.2    Chemical name     TLC      ([α].sub.D)                  __________________________________________________________________________    3(13)                                                                               ##STR91## N-(3-mercapto-2S-benzylpropionyl)- L-glutamic acid                            α-(2-benzthiazolyl) amide                                                                 Rf 0.25 (acetic acid: chloroform =                                            5:95)    +9.83° (CHCl.sub.3, c =                                                1.005)                             3(14)                                                                               ##STR92## N-(3-mercapto-2R-benzylpropionyl)- L-glutamic acid                            α-(2-benzthiazolyl) amide                                                                 Rf 0.29 (chloroform: tetrahydrofuran:                                         acetic acid = 30:8:1)                                                                  -35.6° (CHCl.sub.3, c =                                                 1.00)                             __________________________________________________________________________

EXAMPLE 4 N-(3-mercapto-2RS-benzylpropionyl)-L-aspartic acidα-(2,3-dihydro-1-methyl-5-phenyl-1H-1,4-benzodiazepin-7-yl)amideβ-methyl ester ##STR93##

To a solution of N-(3-acetylthio-2RS-benzylpropionyl)-L-aspartic acidα-(2,3-dihydro-1-methyl-5-phenyl-IH-1,4-benzodiazepin-7-yl) amideβ-benzyl ester (prepared by the same procedure as a series of reactionsof Reference Example 1 →Reference Example 2 →Reference Example 3, byusing the corresponding starting materials, 1,686 g) in methanol (10 ml)was added potassium carbonate (0.688 g), and the mixture was stirred forone hour at room temperature. The reaction mixture was diluted withmethylene chloride (100 ml), washed with water and a saturated aqueoussolution of sodium chloride, dried over magnesium sulfate andconcentrated under reduced pressure. The residue was purified by columnchromatography on silica gel (methylene chloride :methanol =30 : 1) togive the title compound (0.735 g) as orange amorphous solid having thefollowing physical data:

TLC (methylene chloride : methanol =95 : 5) : Rf 0.37;

NMR: δ6 8.32 and 7.98(1H, s), 7.68˜6.64(14H, m), 4.90˜4.75(IH, m),3.88˜3.40(7H, m), 3.20˜1.92(1OH, m), 1.40(1H, 2xt);

MS:m/z 558(M⁺), 526, 510, 498:

IR(KBr): ν3600˜2500, 1715, 1650, 1610, 1490, 1290, 1180, 690.

The following desired compound was obtained by the same procedure asabove.

(1) N-(3-mercapto-2RS-benzylpropionyl)-L-glutamic acidα-(2,3-dihydro1-methyl-5-phenyl-IH-1,4-benzodiazepin-7-yl)amide γ-methylester ##STR94##

TLC (methanol : methylene chloride =1 : 9) : Rf 0.56;

NMR: δ8.48 and 8.27(IH, s, 5 : 6), 7.70˜6.90(13H, m), 6.70 and 6.52(IH,d), 4.55˜4.28(1H, m), 3.80˜3.50(4H, m), 3.63 and 3.65(3H, s),3.00˜1.84(9H, m), 2.80 and 2.76(3H, s), 1.44 and 1.39(1H, t);

MS:m/z 572(M⁺), 540, 512;

IR: ν3300, 1740, 1650, 1500.

EXAMPLE 5 N-(3-mercapto-2RS-benzylpropionyl)-L-glutamic acidα-(2,3-dihydro-1-methyl-5-phenyl-1H-1,4-benzodiazepin-7-yl)amide##STR95##

The title compound having the following physical data was obtained bythe same procedure as Example 1, by using the methyl ester prepared inExample 4(1).

TLC (methanol : benzene =2 : 8) : Rf 0.2 ;

Melting point : 126.0˜131.0 ° C.

REFERENCE EXAMPLE 4 α-anilino-L-glutamic acid γ-tert-butyl ester##STR96##

Under an atmosphere of hydrogen, a mixture ofN-(benzyloxycarbonyl)-α-anilino-L-glutamic acid γ-tert butyl ester (7.80g, prepared by the same procedure as Reference Example 1 (the amido bondforming reaction using DCC as a condensing agent, was used instead ofthe method using a mixed acid anhydride) by usingN-(benzyloxycarbonyl)-L-glutamic acid γ-tert-butyl ester as startingmaterial), palladiumcarbon (10 % ; 880 mg) and ethyl acetate (130 ml)was stirred for 3 hours at a room temeperature. The reaction mixture wasfiltered, and to the filtrate was added ethyl acetate (20 ml) containing4N hydrogen chloride, and then the mixture was concentrated underreduced pressure to give hydrochloric acid salt of the title compound ascrude product.

EXAMPLE 6 N-(3-benzoylthio-2S-benzylpropionyl)-α-anilino-L-glutamic acidγ-tert-butyl ester ##STR97##

The title compound having the following physical data was obtained bythe same procedure as Reference Example 3, by using the amine compoundprepared in Reference Example 4 and 3-benzoylthio-2S-benzylpropionicacid.

TLC (n-hexane : ethyl acetate =1 : 1) : Rf 0.56.

EXAMPLE 7 N-(3-mercapto-2S-benzylpropionyl)-α-anilino-L-glutamic acidγ-tert-butyl ester ##STR98##

The title compound having the following physical data was obtained bythe same procedure as Example 4, by using the benzoyl compound preparedin Example 6

TLC (n-hexane : ethyl acetate =1 : 1) : Rf 0.53.

EXAMPLE 8 N-(3-mercapto-2S-benzylpropionyl)-α-anilino-L-glutamic acid##STR99##

To a solution of the tert-butyl ester (770 mg) prepared in Example 7, inethyl acetate (3 ml) was added ethyl acetate (2 ml) containing 4Nhydrogen chloride, and the mixture was stirred for two hours at a roomtemperature. The reaction mixture was concentrated under reducedpressure and the residue was purified by column chromatography on silicagel (methylene chloride : ethyl acetate =9 : 1 →4 : 1 →2 : 1 →ethylacetate) to give the title compound (300 mg) having the followingphysical data:

TLC (chloroform : tetrahydrofuran : acetic acid =15 : 4 : 1) : Rf 0.39;

Optical rotation (c =0.895, absolute C₂ H₅ OH) : [α]_(D) -22.2° .

The desired compounds shown in the following Table VII were obtained bythe same procedure as a series of reaction of Reference Example4→Example 6→Example 7→Example 8, by using corresponding startingmaterials.

                                      TABLE VII                                   __________________________________________________________________________    Example                                             Optical rotation                                                              ([α].sub.D)         No.  Structure          Chemical name      TLC      or IR (ν,                                                                  cm.sup.-1)                __________________________________________________________________________    8(1)                                                                                ##STR100##        N-(3-mercapto-2R-benzylpropionyl)- α-anilino                            -L-glutamic acid   Rf 0.30 (acetic                                                               acid: chloroform                                                                       [α].sub.D                                                               -91.72° (c =                                                           1.01, absolute                                                                C.sub.2 H.sub.5 OH)       8(2)                                                                                ##STR101##        N-(3-mercapto-2S-benzylpropionyl)- α-anilino                            -L-aspartic acid   Rf 0.425 (chloroform: tetrahydr                                               ofuran: acetic acid                                                                    [α].sub.D                                                               -28.64° (c =                                                           1.00, CH.sub.3 OH))       8(3)                                                                                ##STR102##        N-(3-mercapto-2R-benzylpropionyl)- α-anilino                            -L-aspartic acid   Rf 0.46 (acetic                                                               acid: chloroform = 5:95)                                                      (twice developing)                                                                     [α].sub.D                                                               -86.0° (c =                                                            0.82, CH.sub.3 OH)        8(4)                                                                                ##STR103##        N-(3-mercapto-2RS-benzylpropionyl)- α-(4-met                            hoxyanilino)-L-aspartic acid (less polar                                                         Rf 0.44 (chloroform: tetrahydro                                               furan: acetic acid                                                                     3290, 3050, 2930,                                                             1705, 1645, 1515,                                                             1410, 1250, 1170,                                                             1030, 825,                __________________________________________________________________________                                                        700                   

EXAMPLE 9 N-(3-acetylthio-2RS-benzylpropionyl)-γ-anilino-L-glutamic acidα-tert-butyl ester ##STR104##

The title compound having the following physical data was obtained bythe same procedure as a series of reactions of Reference Example 1→Reference Example 4 →Reference Example 3, by usingN-(benzyloxycarbonyl)-L-glutamic acid α-tert-butyl ester as startingmaterial.

TLC (ethyl acetate : n-hexane =1 : 1) : Rf 0.42.

EXAMPLE 10 N-(3-acetylthio-2RS-benzylpropionyl)-γ-anilino-L-glutamicacid ##STR105##

The title compound having the following physical data was obtained bythe same procedure as Reference Example 2, by using the tert-butyl esterprepared in Example 9 as starting material.

TLC (ethyl acetate : methanol =9 : 1) : Rf 0.12.

EXAMPLE 11 N-(3-mercapto-2RS-benzylpropionyl)-γ-anilino-L-glutamic acidα-sodium salt ##STR106##

To the acetyl compound (150 mg, prepared in Example 10) in a mixture ofchloroform (1 ml) and acetonitrile (2 ml) was added cysteamine (i.e. H₂N--(CH₂)₂ --SH, 52.2 mg), and the mixture was stirred for 20 minutes at40° C. The reaction mixture was diluted with ethyl acetate, washed withwater, dried over magnesium sulfate and concentrated under reducedpressure. The residue was purified by column chromatography on silicagel (ethyl acetate : n-hexane =1 : 1) to give the free acidcorresponding to the title compound.

The obtained free acid compound was dissolved in a small amount ofmethanol and IN aqueous solution of sodium hydroxide and water wereadded thereto. The mixture was stirred for 5 minutes at roomtemperature. The reaction mixture was lyophilized to give the titlecompound (sodium salt) having the following physical data:

Melting poin : 131.0˜135° C.;

TLC (chloroform : tetrahydrofuran : acetic acid =80 : 15 : 5) : Rf 0.23.

EXAMPLE 12 N-(3-mercapto-2RS-benzylpropionyl)-β-anilino-L-aspartic acidand the corresponding α-sodium salt ##STR107##

The title compound having the following physical data was obtained bythe same procedure as a series of reactions of Reference Example 1→Reference Example 2 →Reference Example 3 →Example 11, by usingN-(tert-butoxycarbonyl) L-aspartic acid α-benzyl ester as startingmaterial.

(1) free acid

TLC (chloroform : tetrahydrofuran : acetic acid =15 : 4 : 1) : Rf 0.26 ;

IR: ν3600˜2300, 1725, 1650, 1600, 1530, 1440, 755, 695 cm⁻¹.

(2) sodium salt

TLC (methylene chloride: tetrahydrofuran : acetic acid =15 : 4 : 1) : Rf0.35;

IR: ν3640˜2400, 1650, 1630, 1595, 1530, 1390, 1380 cm⁻¹.

The desired compounds shown in the following Table VIII were obtained bythe same procedure as Example 12 by using corresponding startingmaterials.

                                      TABLE VIII                                  __________________________________________________________________________     ##STR108##                                                                   Example                                        Optical rotation                                                              ([α].sub.D)              No.  R             Chemical name     TLC       or IR (ν,                   __________________________________________________________________________                                                   cm.sup.-1)                     12(1)                                                                               ##STR109##   N-(3-mercapto-2RS-benzylpropionyl)- β-anilino-L-as                       partic acid α-sodium salt (more polar                                                     Rf 0.31 (chloroform: tetrahydrofuran:                                          acetic acid = 15:4:1)                                                                  [α].sub.D                                                               +22.2° (c = 1.53,                                                      absolute C.sub.2 H.sub.5                                                      OH) (as free acid)             12(2)                                                                               ##STR110##   N-(3-mercapto-2RS-benzylpropionyl)- β-anilino-L-as                       partic acid α-sodium salt (less polar                                                     Rf 0.30 (chloroform: tetrahydrofuran:                                          acetic acid = 15:4:1)                                                                  [α].sub.D                                                               -6.73° (c = 1.54,                                                      absolute C.sub.2 H.sub.5                                                      OH) (as free acid)             12(3)                                                                               ##STR111##   N-(3-mercapto-2RS-benzylpropionyl)- β-(4-fluoroani                       lino)-L-aspartic acid                                                                           Rf 0.36 (chloroform: methanol:                                                acetic acid = 30:5:1)                                                                   3650˜2200(3280),                                                        1715, 1650, 1525, 1500,                                                       1405, 1210, 825                12(4)                                                                               ##STR112##   N-(3-mercapto-2RS-benzylpropionyl)- β-(4-chloroani                       lino)-L-aspartic acid                                                                           Rf 0.38 (chloroform: methanol:                                                acetic acid = 30:5:1)                                                                   3645˜2200(3290),                                                        1715, 1655, 1600, 1530,                                                       1490, 1400                     12(5)                                                                               ##STR113##   N-(3-mercapto-2RS-benzylpropionyl)- β-(4-iodoanili                       no)-L-aspartic acid                                                                             Rf 0.40 (chloroform: methanol:                                                acetic acid = 30:5:1)                                                                   3650˜2200(3280),                                                        1720, 1650, 1590, 1515,                                                       1480, 1400, 810                12(6)                                                                               ##STR114##   N-(3-mercapto-2RS-benzylpropionyl)- β-(4-methoxyan                       ilino)-L-aspartic acid                                                                          Rf 0.38 (chloroform: methanol:                                                acetic acid = 30:3:1)                                                                   3650˜2700, 1640,                                                        1600, 1505, 1410, 1245,                                                       1025, 825                      12(7)                                                                               ##STR115##   N-(3-mercapto-2RS-benzylpropionyl)- β-[4-(N,N-dime                       thylamino)anilino]-L-  aspartic acid                                                            Rf 0.23 (chloroform: methanol:                                                acetic acid = 30:3:1)                                                                   3650˜2200(3280),                                                        1650, 1600, 1510, 1310,                                                       820, 700                       12(8)                                                                               ##STR116##   N-(3-mercapto-2RS-benzylpropionyl)- β-(4-trifluoro                       methylanilino)-L- aspartic acid                                                                 Rf 0.33 (chloroform: methanol:                                                acetic acid = 30:3:1)                                                                   3670˜2250(3280,                                                         3050, 2920), 1720, 1650,                                                      1605, 1525, 1405, 1320,                                                       1155, 1110, 1065, 840          12(9)                                                                               ##STR117##   N-(3-mercapto-2RS-benzylpropionyl)- L-aspartic acid                           β-(2,3-dihydro-1- methyl-5-phenyl-1H-1,4-                                benzodiazepin-7-yl)amide                                                                        Rf 0.15 (methylene chloride:                                                  methanol: acetic acid                                                                   3650˜2400, 1640,                                                        1610, 1520, 1490, 1395,                                                       1295                           __________________________________________________________________________

REFERENCE EXAMPLE 5N-(3-mercapto-2RS-benzylpropionyl)-α-anilino-L-glutamic acidγ-tert-butyl-diphenylsilyl ester ##STR118##

To a solution of the carboxylic acid (1.69 g, prepared in Example 1) indimethylformamide (12 ml) were added imidazole (0.631 g) andsuccessively tert-butyldiphenylsilyl chloride (1.23 ml) at a roomtemperature, and the mixture was stirred for 3 hours at the sametemperature. The reaction mixture was dissolved in ether (150 ml),washed with 1N hydrochloric acid, 1N aqueous solution of sodiumhydroxide and water, successivly, dried over magnesium sulfate andconcentrated under reduced pressure. The residue was purified by columnchromatography on silica gel (ethyl acetate: methylene chloride =5 : 95→1 : 9) to give the title compound (2.37 g) as white powder having thefollowing physical data:

TLC (ethyl acetate : methylene chloride : 1 : 9) : Rf 0.48 ;

NMR: δ8.58 and 8.46(1H, s×2), 7.85˜6.98(20H, m), 6.84˜6.62(1H, d×2),4.56˜4.39(1H, m), 2.98˜1.02(19H, m);

MS:m/z 638(M⁺), 581.

REFERENCE EXAMPLE 6N-(3-nicotinoylthio-2RS-benzylpropionyl)-α-anilino-L-glutamic acidγ-tert-butyl-diphenylsilyl ester ##STR119##

To a mixture of the mercapto compound (1.43 g, prepared in ReferenceExample 5), nicotinic acid (0.276 g) and diphenylphosphoryl azide (0.99ml) and dimethylformamide (5 ml), was added triethylamine (0.63 ml) at0° C. and the mixture was stirred for 3 hours at a room temperature. Tothe reaction mixture was added water (30 ml) and the mixture wasextracted with ethyl acetate (80 ml ×2). The extract was dried overmagnesium sulfate and concentrated under reduced pressure. The residuewas purified by column chromatography on silica gel (methylene chloride: ethyl acetate =3 : 1→2 : 1) to give the title compound (0.35 g) aswhite powder having the following physical data:

TLC (methylene chloride : ethyl acetate =2 : 1) : Rf 0.44 and 0.38;

NMR: δ9.12 and 9.02(1H, dd×2), 8.73 and 8.70 (IH, dd ×2), 8.53 and8.45(IH, s ×2), 8.13 and 7.95(IH, ddd ×2), 7.75˜7.00(21H, m),6.60˜6.42(1H, d×2), 4.52˜4.37(IH, m), 3.45˜1.50(9H, m), 1.11˜1.08(9H, s×2) ;

MS:m/z 685, 547.

REFERENCE EXAMPLE 13N-(3-nicotinoylthio-2RS-benzylpropionyl)-α-anilino-L-glutamic acid##STR120##

A solution of the ester compound (350 mg, prepared in Reference Example6) in a mixture of acetic acid (3 ml), tetrahydrofuran (1 ml) and water(1 ml) was stirred for 16 hours at a room temperature, and the reactionmixture was concentrated under reduced pressure. The residue waspurified by column chromatography on silica gel (chloroform : methanol :acetic acid =80 : 3 : 1 →50 : 3 : 1) to give the title compound (175 mg)as white powder having the following physical data:

TLC (chloroform : methanol : acetic acid =50 : 3 : 1) : Rf 0.47;

NMR(CDCl₃ +DMSO - d₆) : δ9.68 and 9.62(1H, s ×2), 9.05 and 9.00 (1H, d×2), 8.77 and 8.73(1H, dd ×2), 8.34˜8.04(2H, m), 7.60˜6.96(11H, m),4.62˜4.36(1H, m), 3.80˜1.60(9H, m);

MS:m/z 487, 348, 335;

IR(KBr): ν3600˜2100 (3370, 3040, 2925), 1705, 1640, 1635, 1590, 1525,1440, 1210, 915, 690 cm⁻¹.

The desired compounds shown in the following Table IX and X wereobtained by the same procedure as a series of reactions of ReferenceExample 5 →Reference Example 6 →Example 13, by using correspondingstarting materials.

                                      TABLE IX                                    __________________________________________________________________________                                                          Optical rotation        Example                                               ([α].sub.D)                                                             or                      No.  Structure                 Chemical name TLC      IR (ν,               __________________________________________________________________________                                                          cm.sup.-1)              13(1)                                                                               ##STR121##               N-(3-nicotinoylthio-2RS- benzylpropionyl)-.                                   beta.-anilino-L- aspartic                                                                   Rf 0.37 (chloroform:                                                          methanol: acetic acid =                                                       30:5:1)  3650˜2200(3275                                                          , 3030), 1715,                                                                1660, 1595, 1530,                                                             1435, 1200, 910,                                                              755, 690                13(2)                                                                               ##STR122##               N-(3-nicotinoylthio-2S- benzylpropionyl)-L-                                   glutamic acid α-(2-benzthia-                                            zolyl)amide.acetic acid salt                                                                Rf 0.17 (acetic acid:                                                         chloroform                                                                             [α].sub.D                                                               -38.6° (c =                                                            0.975, tetrahydrofur                                                          an)                     13(3)                                                                               ##STR123##               N-(3-nicotinoylthio-2R- benzylpropionyl)-.a                                   lpha.-anilino-L- glutamic                                                     acid.hydrochloric acid salt                                                                 Rf 0.53 (methylene chloride:                                                  etrahydrofuran: acetic acid                                                   = 7:2:1) 3270,                                                                         1705 (shoulder),                                                              1665, 1598, 1530,                                                             1442, 1214, 930,                                                              760, 702                __________________________________________________________________________

                                      TABLE X                                     __________________________________________________________________________     ##STR124##                                                                   Example                                                                       No.   R                  Chemical name     TLC       Optical                  __________________________________________________________________________                                                         rotation                 13(4)                                                                                ##STR125##        N-(3-nicotinoylthio-2S- benzylpropionyl)-α-                             anilino-L- glutamic acid.hydrochloric acid                                                      Rf 0.32 (acetic                                                               acid: chloroform                                                                        [α].sub.D                                                               -58.6° (c =                                                            1.055, CH.sub.3 OH)      13(5)                                                                                ##STR126##        N-(3-diphenylacetylthio-2S- benzylpropionyl)-.alp                             ha.-anilino-L- glutamic acid                                                                    Rf 0.40 (methanol: chloroform                                                 = 10:90)  [α].sub.D                                                               -52.9° (c =                                                            1.035, CHCl.sub.3)       13(6)                                                                                ##STR127##        N-(3-isonicotinoylthio-2S- benzylpropionyl)-.alph                             a.-anilino-L- glutamic acid                                                                     Rf 0.36 (chloroform: tetrahydro                                               furan: acetic acid                                                                      [α].sub.D                                                               -77.48° (c =                                                           1.00, CH.sub.3 OH)       13(7)                                                                                ##STR128##        N-[3-(2,2-dimethyl-1,3-dioxolan-4S- carbonyl)thio                             -2S-benzylpropionyl]- α-anilino-L-glutamic                              acid              Rf 0.25 (ethyl                                                                          [α].sub.D                                                               -58.80° (c =                                                           1.085, CHCl.sub.3)       13(8)                                                                                ##STR129##        N-[3-[N-(9-fluorenyl)succinamoyl] thio-2S-benzylp                             ropionyl]-α-anilino- L-glutamic                                                           Rf 0.42 (actic                                                                acid: chloroform:                                                                       [α].sub.D                                                               -127.5° (c =                                                           1.00, dimethyl                                                                sulfoxide)               __________________________________________________________________________

EXAMPLE 14 N-(3-nicotinoylthio-2S-benzylpropionyl)-α-anilino-L-asparticacid hydrochloride ##STR130##

The title compound having the following physical data was obtained bythe same procedure as a series of reactions of Reference Example 1 (theamido bond forming reaction using DCC as a condensing agent, was usedinstead of the method using a mixed acid anhydride)→Reference Example 4→Example 6 →Example 7 Reference Example 6 →Example 8, by usingN-(benzyloxycarbonyl)-L-aspartic acid β-tert-butyl esterdicyclohexylamine salt as starting material.

TLC (methylene chloride : tetrahydrofuran : acetic acid =7 : 2 : 1) : Rf0.56 ;

Optical rotation (c =1.00, CH₃ OH) : [α]_(D) -66.22°.

EXAMPLE 15 N-(3-benzoylthio-2S-benzylpropionyl)-α-anilino-L-asparticacid ##STR131##

The title compound having the following physical data was obtained bythe same procedure as a series of reactions of Reference Example 1 (theamido bond forming reaction using DCC as a condensing agent, was usedinstead of the method using a mixed acid anhydride)→Reference Example 4→Example 6 →Example 10, by using N-(benzyloxycarbonyl)-L-aspartic acidβ-tert-butyl ester dicyclohexylamine salt as starting material.

TLC (chloroform : acetic acid =95 : 5) : Rf 0.31;

IR : ν3290, 3040, 1700, 1645, 1600, 1520, 1440, 1200, 1170, 910, 755,690 cm⁻¹.

The following desired compound was obtained by the same procedure asabove.

(1) N-(3-benzoylthio-2S-benzylpropionyl)-α-anilino-L-glutamic acid##STR132##

TLC (chloroform:acetic acid=95:5):Rf 0.29;

IR:ν3270, 3050, 1705, 1655, 1640, 1600, 1530, 1490, 1445, 1205, 910,750, 685 cm⁻¹.

REFERENCE EXAMPLE 7N-[3-(phthalid-3-yl)thio-2S-benzylpropionyl]-α-anilino-L-glutamic acidγ-tert-butyldiphenylsilyl ester ##STR133##

To a solution of the thioether compound (178 mg, prepared by the sameprocedure as Reference Example 5 by using the compound prepared inExample 8 as starting material) in acetone (2 ml) were added3-phthalidyl chloride (56.1 mg) and potassium carbonate (46 mg) at roomtemperature, and the mixture was stirred for 2 hours at the sametemperature. The reaction mixture was diluted with a mixture of ethylacetate and ether, washed with water and a saturated aqueous solution ofsodium chloride, successively, dried over sodium sulfate andconcentrated under reduced pressure. The residue was purified by columnchromatography on silica gel (ethyl acetate: n-hexane =1 : 2) to givethe title compound (153.6 mg) having the following physical data :

TLC (ethyl acetate : n-hexane =1 : 2 ) : Rf 0.2.

EXAMPLE 16N-3-(phthalid-3-yl)thio-2S-benzylpropionyl-α-anilino-L-glutamic acid##STR134##

The title compound having the following physical data was obtained bythe same procedure as Example 13, by using the silyl ester prepared inReference Example 7 as starting material.

TLC (chloroform : acetic acid =95 : 5) : Rf 0.40;

IR : ν3255, 1750, 1640, 1600, 1520, 1440 cm⁻¹.

The following desired compounds were obtained by the same procedure asabove.

(1) N-[3-(phthalid-3-yl)thio-2S-benzylpropionyl]-α-anilino-L-asparticacid ##STR135##

TLC (chloroform : tetrahydrofuran : acetic acid =15 : 4 : 1) : Rf 0.51;

IR : ν3280, 3030, 1760, 1640, 1600, 1525, 1440, 1285, 1170, 940, 760,725, 695 cm⁻¹.

(2) N-3-(phthalid-3-yl)thio-2S-benzylpropionyl-L-glutamic acidα-(2-benzthiazolyl)amide ##STR136##

TLC (chloroform : acetic acid =98 : 2) : Rf 0.25;

IR : ν3300, 1760, 1640, 1600, 1540, 1440 cm⁻¹.

EXAMPLE 17N-[3-(phthalid-3R(or-3S)-yl)thio-2S-benzylpropionyl]-α-anilino-L-glutamicacid ##STR137##

The diastereomer (i.e. 3RS-mixture, prepared in Example 16) wasrecrystallized twice from ethyl acetate to give the 3R-isomer having thefollowing physical data. Further, mother liquor obtained in theseparation of R-isomer, was concentrated under reduced pressure and theresidue was dissolved in ethanol and recrystallized twice from ethanolto give the 3S-isomer having the following physical data.

(1) 3R-isomer

TLC (methylene chloride : methanol =9 : 1) : Rf 0.14;

IR : ν3270, 3060, 1758, 1732, 1671, 1646, 1531, 1446, 1295, 1177, 960,756, 697 cm⁻¹.

(2) 3S-isomer

Melting point: 174° ˜176° C.;

TLC (methylene chloride : methanol =9 : 1) : Rf 0.12;

IR : ν3286, 3060, 1774, 1709, 1677, 1639, 1533, 1445, 1290, 953, 726,701 cm⁻¹.

FORMULATION EXAMPLE

The following components were admixed in conventional method and punchedout to obtain 100 tablets each containing 50 mg of active ingredient.

    ______________________________________                                        N-(3-mercapto-2RS-benzylpropionyl)-                                                                  5.0 g                                                  α-anilino-L-glutamic acid                                               Cellulose calcium glycolate                                                                          0.2 g                                                  (disinintegrating agent)                                                      Magnesium stearate     0.1 g                                                  (lubricating agent)                                                           Microcrystaline cellulose                                                                            4.7 g                                                  ______________________________________                                    

What is claimed is:
 1. An amino acid derivative which is N-(3-mercapto-2RS-benzylpropionyl)-L-glutamic acid α-(4-pyridyl)amide.
 2. A pharmaceutical composition for the prevention and treatment of pain, anxiety or convulsion, which comprises, as the active ingredient, an effective amount of an amino acid derivative as claimed in claim 1 or a non-toxic salt thereof and a pharmaceutically acceptable carrier.
 3. A method for the prevention and treatment of pain, anxiety or convulsion, which comprises, administering an effective amount of an amino acid derivative as claimed in claim 1 or a non-toxic salt thereof. 